The protonophore CCCP interferes with lysosomal degradation of autophagic cargo in yeast and mammalian cells.

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The protonophore CCCP interferes with lysosomal degradation of autophagic cargo in yeast and mammalian cells ab

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Benjamin S Padman , Markus Bach , Giuseppe Lucarelli , Mark Prescott & Georg Ramm

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Department of Biochemistry and Molecular Biology; Monash University; Clayton campus; Victoria, Australia b

Monash Micro Imaging; Monash University; Clayton campus; Victoria, Australia Published online: 27 Sep 2013.

To cite this article: Benjamin S Padman, Markus Bach, Giuseppe Lucarelli, Mark Prescott & Georg Ramm (2013) The protonophore CCCP interferes with lysosomal degradation of autophagic cargo in yeast and mammalian cells, Autophagy, 9:11, 1862-1875, DOI: 10.4161/auto.26557 To link to this article: http://dx.doi.org/10.4161/auto.26557

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Basic Research Paper

Autophagy 9:11, 1862–1875; November 2013; © 2013 Landes Bioscience

The protonophore CCCP interferes with lysosomal degradation of autophagic cargo in yeast and mammalian cells Benjamin S Padman,1,2 Markus Bach,1,2 Giuseppe Lucarelli,1 Mark Prescott,1 and Georg Ramm1,2,* Department of Biochemistry and Molecular Biology; Monash University; Clayton campus; Victoria, Australia; 2Monash Micro Imaging; Monash University; Clayton Campus; Victoria, Australia

Downloaded by [University of Louisville] at 05:43 29 December 2014

Keywords: CCCP, DiOC6, TMRM, MitoTracker, lysosomal chloride, mitophagy, Parkin, PARK2 Abbreviations: AntiA, antimycin A; Baf, bafilomycin A1; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CLEM, correlative light and electron microscopy; DiOC6, 3,3´-dihexyloxacarbocyanine iodide; LTG, LysoTracker Green DND-26; LTR, LysoTracker Red DND-99; MTG, MitoTracker Green FM; MTR, MitoTracker Red FM; Oligo, oligomycin; TEM, transmission electron microscopy; TMRM, tetramethylrhodamine methyl ester; WM, wortmannin

Mitophagy is a selective pathway, which targets and delivers mitochondria to the lysosomes for degradation. Depolarization of mitochondria by the protonophore CCCP is a strategy increasingly used to experimentally trigger not only mitophagy, but also bulk autophagy. Using live-cell fluorescence microscopy we found that treatment of HeLa cells with CCCP caused redistribution of mitochondrially targeted dyes, including DiOC6, TMRM, MTR, and MTG, from mitochondria to the cytosol, and subsequently to lysosomal compartments. Localization of mitochondrial dyes to lysosomal compartments was caused by retargeting of the dye, rather than delivery of mitochondrial components to the lysosome. We showed that CCCP interfered with lysosomal function and autophagosomal degradation in both yeast and mammalian cells, inhibited starvation-induced mitophagy in mammalian cells, and blocked the induction of mitophagy in yeast cells. PARK2/Parkin-expressing mammalian cells treated with CCCP have been reported to undergo high levels of mitophagy and clearance of all mitochondria during extensive treatment with CCCP. Using correlative light and electron microscopy in PARK2-expressing HeLa cells, we showed that mitochondrial remnants remained present in the cell after 24 h of CCCP treatment, although they were no longer easily identifiable as such due to morphological alterations. Our results showed that CCCP inhibits autophagy at both the initiation and lysosomal degradation stages. In addition, our data demonstrated that caution should be taken when using organelle-specific dyes in conjunction with strategies affecting membrane potential.

Introduction Lysosomes play an essential role in cellular homeostasis by acting as an end point for the degradation of intracellular and extracellular structures by a panel of hydrolases contained within their acidic lumen.1 The low pH of the lysosomal lumen is maintained by a vacuolar-type H+ -ATPase (V-ATPase). Pharmacological inhibition of the V-ATPase by bafilomycin A1 (Baf) results in alkalinization of lysosomal pH and attenuation of lysosomal degradation. Several membrane trafficking pathways intersect with the lysosome including endocytosis, phagocytosis, and autophagy. Mammalian macroautophagy (referred to here as autophagy) is a multistep trafficking pathway. Upon initiation of autophagy, cytoplasmic cargo is sequestered within double-membraned vesicles called autophagosomes, which fuse with lysosomes to degrade

the cargo.2 Assays of autophagy typically rely on the autophagy marker LC3/ATG8, which is recruited to autophagosomes by a conjugation system that shares similarities with the ubiquitination machinery.3 Mitophagy is a form of selective autophagy that targets mitochondria for degradation. The PINK1-PARK2/Parkin model of mitophagy is a widely studied system in mammalian cells, which can be experimentally initiated by exposure of PARK2-expressing cells to carbonyl cyanide m-chlorophenylhydrazone (CCCP).4 CCCP is a protonophore capable of increasing membrane proton conductance by several orders of magnitude,5 thereby causing mitochondrial depolarization and uncoupling of respiration.6 Under normal conditions, PINK1 is imported to the mitochondrial intermembrane space via a membrane-potential dependent process, where it is proteolytically degraded.7,8 Depolarization of mitochondria blocks the import of PINK1, which subsequently

*Correspondence to: Georg Ramm; Email: [email protected] Submitted: 05/02/2013; Revised: 09/17/2013; Accepted: 09/20/2013 http://dx.doi.org/10.4161/auto.26557 1862 Autophagy

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accumulates on the outer mitochondrial membrane and recruits the E3 ubiquitin ligase PARK2.9 Ubiquitination of key mitochondrial membrane proteins then targets the mitochondria for degradation by autophagy and the proteasome.10 CCCP is increasingly being used to investigate pathways independent of the PINK1-PARK2 model.11 In a recent study reporting the effects of CCCP in the absence of PARK2,12 Ding et al. found that mitochondria treated with CCCP form “spheroid structures” that interact with acidic compartments. These findings were based on observations made using fluorescence microscopy, which revealed colocalization between MitoTracker Green FM (MTG) and lysosomal markers in the presence of CCCP. Our investigations into the effects of CCCP showed that lysosomal markers colocalize with mitochondrial dyes, but not with mitochondria. CCCP treatment of HeLa cells stained with one of either TMRM (tetramethylrhodamine methyl ester), DiOC6 (3,3´-dihexyloxacarbocyanine iodide), MTR (MitoTracker Red FM) or MTG resulted in localization of the mitochondrial dye to small, highly mobile vesicles, which we concluded are lysosomes. CCCP-induced translocation of cationic dyes to the lysosome was inhibited in cells treated with Baf and was absent when antimycin A (AntiA) and oligomycin (Oligo) were used to specifically depolarize mitochondria, suggesting that the effect was dependent on rapid proton efflux from the lysosomes caused by CCCP. After establishing the effect of CCCP on lysosomal membrane potential, we have investigated the consequences of these changes on lysosomal function. While effects of CCCP on cellular organelles including the lysosome are known,13 these have not been addressed in the context of autophagy or the PINK1PARK2 model of mitophagy.14 Consistent with the requirement of low lysosomal pH for degradation, we found that CCCP prevents lysosomal degradation, and that autophagic degradation in yeast and mammalian cells is modulated dependent on the extracellular pH.

Results Treatment of cells with CCCP results in relocalization of mitochondrial dyes to lysosomes HeLa cells were stained with the potentiometric carbocyanine dye DiOC6 and the effect of CCCP addition on the labeling of mitochondria was observed using live-cell fluorescence microscopy. In the absence of CCCP, green fluorescence emission of DiOC6 was localized to a reticular network

distributed throughout the cell in a manner typical for mitochondria (Fig. 1A). Addition of 20 µM CCCP to DiOC6-labeled cells initiated a rapid loss of mitochondrial DiOC6 fluorescence within 5 s, and coincided with an increase in cytosolic DiOC6 fluorescence emission. After a further 15 s of incubation with CCCP, green fluorescence localized to small puncta. Live-cell imaging indicated that these puncta were highly mobile, and were undetectable prior to the addition of CCCP. These results indicate that CCCP-induced loss of mitochondrial membrane potential results in efflux of DiOC6 from the mitochondria into the cytoplasm and subsequent localization to punctate structures. We sought to investigate the identity of the punctate structures. HeLa cells expressing a red fluorescent protein (DsRed2Mito) targeted to the mitochondrial matrix were stained with DiOC6, and imaged using live-cell fluorescence microscopy (Fig. 1B). CCCP treatment resulted in a rapid loss of DiOC6 from the mitochondrial reticulum, whereas the distribution of DsRed2-Mito labeling was similar to that observed in cells not treated with CCCP. Green DiOC6-positive puncta were observed after a further 20 s of CCCP incubation. These puncta were not labeled with red fluorescence of dsRed2-Mito suggesting that the puncta are not of mitochondrial origin. To identify the DiOC6-labeled puncta we utilized a number of different organelle-specific probes (not shown). HeLa cells labeled with both LysoTracker Red DND-99 (LTR) and DiOC6 displayed colocalization of the dyes within 30 s of CCCP treatment (Fig. 1C; Video S1), which remained evident 1 h after the addition of CCCP (Fig. 1D and E). In contrast, HeLa cells expressing DsRed2-Mito, stained with LysoTracker Green DND-26 (LTG) and incubated with CCCP for 1 h exhibited reticular and toroidal DsRed2-Mito-labeled structures, which were negative for LTG staining (Fig. 1L and M). These results show that DiOC6 labeling is segregated from mitochondria, and that DiOC6 localizes to lysosomal compartments upon CCCP treatment. We then tested whether other commonly used mitochondrial dyes localize to lysosomes in the presence of CCCP. HeLa cells were either stained with the red mitochondrial dyes TMRM or MTR and counterstained with LTG, or stained with MTG and counterstained with LTR. Each of the mitochondrial dyes tested colocalized with LysoTracker dyes after 1 h of incubation with CCCP (Fig. 1F–K). To ensure that each mitochondrial marker was genuinely targeted to the mitochondria of untreated cells, we verified their colocalization in the absence of CCCP (Fig. 1O–R). In a separate experiment, we observed localization

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Figure 1 (See previous page). CCCP causes retargeting of mitochondrial dyes to the lysosome. (A) HeLa cells were stained with 20 nM DiOC6 for 30 min before treatment with 20 µM CCCP during live-cell acquisition of time-series images via fluorescence microscopy. (B) HeLa cells transiently expressing dsRed2-Mito were stained with 20 nM DiOC6 and treated with 20 µM CCCP during live-cell fluorescence microscopy as in (A). (C) HeLa cells were stained with 20 nM DiOC6 and 100 nM LTR before treatment with 20 µM CCCP during live-cell fluorescence microscopy as in (A). (D–K) HeLa cells were stained for 30 min with (D and E) 20 nM DiOC6 and 100 nM LTR; (F and G) 500 nM TMRM and 100 nM LTG; (H and I) 100 nM MTG and 100 nM LTR; or (J and K) 100 nM MTR and 100 nM LTG, then imaged by live-cell fluorescence microscopy (D, F, H, and J) prior to and (E, G, I, and K) after incubation with 20 µM CCCP for 1 h. (L and M) HeLa cells transiently expressing dsRed2-Mito were stained with 100 nM LTG for 30 min, then imaged by live-cell fluorescence microscopy (L) before and (M) after incubation with 20 µM CCCP for 1 h. (O–R) HeLa cells or (P and R) HeLa cells transiently expressing dsRed2-Mito were stained for 30 min with (O) 20 nM DiOC6 and 500 nM TMRM; (P) 20 nM DiOC6; (Q) 100 nM MTG and 100 nM MTR; or (R) 100 nM MTG, then imaged by live-cell fluorescence microscopy. (S–V) HeLa cells transiently expressing dsRed2-Mito were incubated with 20 µM CCCP for 1 h (S and U) before imaging, then stained for 1 h with (T) 20 nM DiOC6 or (V) 100 nM MTG for 1 h. Scale bars: 10 µm. n > 3.

of mitochondrial dyes to lysosomes when HeLa cells were preincubated with CCCP for 1 h before staining with DiOC6 or MTG (Fig. 1S–V). Collectively, these results suggest that CCCP promotes lysosomal accumulation of mitochondrial dyes. Prompted by our data, we formulated a theory to explain the accumulation of mitochondrial dyes in lysosomal compartments (Fig. S4A). The mitochondrial proton gradient generates a negative membrane potential, which promotes the accumulation of positively charged (cationic) dyes such as DiOC6, TMRM, MTG and MTR.15,16 CCCP collapses the mitochondrial proton gradient by rapidly transporting protons across lipid bilayers, thereby removing the negative membrane potential that drives the accumulation of cationic dyes.5 In contrast, lysosomes generate a proton gradient across their membranes during their acidification by transporting protons into the lysosomal lumen by the action of the V-ATPase.17 This proton gradient generates an opposing potential difference, which lysosomes counter-balance by exporting cations,18 and by importing chloride via Cl- /H+ antiporters.19 Given that CCCP only transports protons, we hypothesized that it may promote collapse of the lysosomal pH gradient without immediately altering ion counter-flux or counter-gradients, thereby generating a negative membrane potential capable of driving the accumulation of cationic mitochondrial dyes. To test this hypothesis, we treated HeLa cells with 100 nM Baf to inhibit lysosomal acidification and H+ -driven Cl- import.13 HeLa cells were pre-incubated with Baf for 3 h before staining with TMRM and LTG, and then incubated a further 1 h in the presence of Baf and CCCP (Fig. 2A and B). Neither TMRM- nor

LTG-positive puncta were observed in the cells. The results indicate that CCCP generates a lysosomal membrane potential by dissipating the pH gradient in the presence of the ion countergradients formed during acidification of the lumen. We further tested our hypothesis by treating HeLa cells with the specific mitochondrial inhibitors AntiA (complex III inhibitor) and Oligo (F1Fo ATP synthase inhibitor) in combination, to selectively depolarize mitochondria without dissipating the lysosomal pH-gradient. HeLa cells were stained with either TMRM and LTG (Fig. 2C) or DiOC6 and LTR (Fig. 2E) before incubation with 1 µM AntiA and 1 µM Oligo for 1 h. Incubation of cells with a combination of AntiA and Oligo resulted in cytosolic labeling by TMRM and DiOC6, and no labeling of puncta with LTG or LTR (Fig. 2D and F). Subsequent treatment of these cells with 20 µM CCCP promoted the labeling of lysosomes by the cationic fluorophores (Fig. 2G; Video S2). In a separate experiment, depolarization of individual mitochondria (and not lysosomes) by photo-irradiation did not result in accumulation of cationic fluorophores in lysosomes (data not shown). These results strongly support the notion that translocation of cationic mitochondrial dyes to the lysosome in CCCP-treated cells is promoted by collapse of the lysosomal proton gradient, independently of mitochondrial depolarization. CCCP blocks lysosomal degradation of endocytic and autophagic cargo, and inhibits the induction of autophagy in mammalian cells An acidic lysosomal lumen is required for the optimal function of resident hydrolases. We therefore investigated whether CCCP




Figure 2. CCCP generates a lysosomal membrane potential, which accumulates mitochondrial dyes. (A and B) HeLa cells were incubated with 100 nM Baf for 3 h before staining with 500 nM TMRM and 100 nM LTG and imaging by live-cell fluorescence microscopy, (A) before and (B) after incubation with 20 µM CCCP for 1 h. (C–F) HeLa cells were stained for 30 min with (C and D) 500 nM TMRM and 100 nM LTG, or (E and F) 20 nM DiOC6 and 100 nM LTR, then imaged by live-cell fluorescence microscopy (C and E) before and (D and F) after incubation with 1 µM Oligo and 1 µM AntiA for 1 h. (G) HeLa cells were stained with 20 nM DiOC6 and 100 nM LTR for 30 min and incubated with 1 µM Oligo and 1 µM AntiA for 1 h before treatment with 20 µM CCCP during live-cell acquisition of time-series images via fluorescence microscopy. Scale bars: 10 µm. n > 3.

of AntiA and Oligo instead of CCCP (Fig. 3E). We found that GFP-LC3 puncta were cleared under these conditions, consistent with the notion that degradation of preformed autophagosomes was inhibited by the action of CCCP on the lysosome. Unexpectedly, cells treated for 2 h with CCCP alone after 4 h starvation did not exhibit an increase in GFP-LC3 puncta or LC3-II/LC3-I ratio (Fig. 3B and D). In contrast Baf, which also blocks the degradation step, led to an increase in LC3, implying that CCCP might affect other steps in the autophagy pathway. To explore the possibility that CCCP inhibits the initiation of autophagy, GFP-LC3 HEK293-A cells were incubated for 2 h with different concentrations of CCCP in either replete culture medium (DMEM+FCS) or during starvation in HBSS (Fig. 3F). CCCP treatment of HBSS-starved cells resulted in fewer GFP-LC3 puncta per cell at all concentrations used, while cells cultured in DMEM+FCS exhibited a slight increase in the number of puncta when treated with 20 µM CCCP. Collectively, these results indicate that CCCP inhibits not only lysosomal function and autophagic degradation, but also the induction of autophagy during starvation. CCCP equilibrates vacuolar pH and blocks the induction of autophagy in yeast cells Reports in the literature conclude that CCCP does not induce mitophagy in yeast cells.23,24 Therefore, we tested the idea that CCCP might be unable to induce mitophagy in yeast cells due to a simultaneous inhibition of autophagy. Yeast cells expressing a mitochondrially targeted pH-biosensor (mtRosella; described in refs. 25,26) were used to monitor the delivery of mitochondria to the acidic yeast vacuole during mitophagy (Fig. 4A). mtRosella is a dual-wavelength emission biosensor consisting of two fluorescent proteins in tandem; a green fluorescent protein whose fluorescence emission is quenched below pH 6.0, and a red fluorescent protein capable of stable fluorescence over a wide pH range, including within the acidic environment of the yeast vacuole. Under growing conditions, the mitochondria in cells expressing mtRosella appear yellow due to the combined green and red fluorescence emission of mtRosella (Fig. 4B). The vacuoles in cells cultured in nitrogen-starvation medium for 24 h (Fig. 4C) show red but not green fluorescence, indicating the delivery of mtRosella-labeled mitochondria to the acidic vacuole. When nitrogen-starved cells were transferred to pH 8.0-buffered medium containing CCCP (5 µM), the vacuoles showed both red and green fluorescence (Fig. 4D), indicating dequenching of the pH-sensitive GFP and revealing the presence of both fluorescent proteins within the vacuole. Addition of CCCP (5 µM) to cells in unbuffered starvation medium (typically pH 4.7)27 resulted in quenching of green fluorescence in mitochondria while vacuoles and mitochondria remained red (not shown). Collectively, these results indicate that CCCP equilibrates the yeast vacuolar pH with the pH of the extracellular medium. We then tested the effect of CCCP on the induction of mitophagy in yeast cells. Fluorescence microscopy was used to observe mtRosella-expressing yeast cells subjected to nitrogen starvation in the presence or absence of CCCP (Fig. 4E and F). The results show that red fluorescence did not accumulate in the

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impairs lysosomal function. We first analyzed the rate at which an endocytosed exogenous marker protein was degraded. HeLa cells were allowed to internalize unconjugated horseradish peroxidase (HRP) for 2 h, after which external HRP was removed by rinsing, and the cells were then cultured in the presence or absence of CCCP (Fig. 3A). Cells were lysed in RIPA buffer and subjected to SDS-PAGE, then immunoblotted for HRP and ACTB/actin, β. The amount of internalized HRP in cells not exposed to CCCP decreased significantly over a period of 24 h. In contrast, the HRP to ACTB ratio in cells treated with CCCP for 24 h remained relatively unchanged. These results indicate that CCCP impairs the degradation of endocytosed exogenous protein. CCCP is increasingly being used to trigger bulk autophagy,11,20 a process which requires functional lysosomes for completion.21 Therefore, we investigated the effects of CCCP on autophagic flux by following the turnover of autophagosomes in cells stably expressing GFP-LC3, which is a marker of autophagosomal membranes. We starved HEK293-A cells expressing GFP-LC3 (GFP-LC3 HEK293-A) for 4 h to induce autophagy, then blocked the formation of new autophagosomes using wortmannin [WM; autophagy and PIK3CA/phosphatidylinositol-4,5-bisphosphatase 3-kinase, catalytic subunit α (class I PI3kinase) inhibitor], effectively “pulse-labeling” the cells with GFP-LC3 positive autophagosomes. After 2 h we quantified the number of remaining autophagosomes (Fig. 3B). GFP-LC3-positive puncta were not observed in samples treated with WM alone, indicating that autophagosomes formed during the 4 h starvation had been degraded. In contrast, cells treated with both WM and CCCP did not show a reduction in the number of autophagosomes. To confirm the effect of CCCP on autophagic flux we quantified the ratio between endogenous LC3-II and its precursor LC3I. HeLa cells were starved for 4 h, followed by 2 h of WM with and without CCCP, lysed in RIPA buffer and proteins resolved by SDS-PAGE to immunoblot for endogenous LC3 (Fig. 3D). We found a significant reduction in the LC3-II/LC3-I ratio in the lysates of cells treated with WM alone, but no such reduction in cells treated with both WM and CCCP. The LC3-II/ LC3-I ratio was reduced in cells treated with WM and Baf due to temporary retention of lysosomal acidity in the presence of Baf,22 which enables lysosomal degradation to continue until the pH gradient has dissipated. In contrast, CCCP rapidly dissipates the lysosomal pH gradient, leading to a complete block in degradation of GPF-LC3-positive autophagosomes. Collectively, these results indicate that CCCP inhibits the clearance of autophagosomes and decreases autophagic flux. To ensure that WM blocks any activation of autophagy by CCCP, we pre-treated GFP-LC3 HEK293-A cells with WM for 30 min before incubating the cells for 90 min with WM in the presence or absence of CCCP, and quantified the number GFP-LC3 positive puncta per cell (Fig. 3C). There was no change in the number of autophagosomes per cell, verifying that CCCP does not induce PIK3CA-independent autophagy. To determine whether the effects of CCCP on autophagic flux were caused by mitochondrial depolarization, we followed the turnover of autophagosomes in the presence of a combination



Figure 3. CCCP inhibits lysosomal degradation and autophagosomal clearance. (A) HeLa cells were loaded with unconjugated HRP, then incubated for 0, 6, 12, and 24 h in the presence or absence of 20 µM CCCP and lysis in RIPA buffer and anti-HRP immunoblotting after SDS-PAGE. HRP was quantified and normalized to the ACTB signal obtained by reprobing the blot with anti-ACTB antibody. (B) GFP-LC3 HEK293-A cells were starved with HBSS for 4 h to induce autophagy, before a 2 h incubation in the presence or absence 1 µM WM, 20 µM CCCP, 100 nM Baf, or a combination thereof. The number of GFP-LC3 puncta per cell was quantified by fluorescence microscopy after fixation of the GFP-LC3 HEK293-A cells. (C) GFP-LC3 HEK293-A cells were incubated in DMEM+FCS with or without 1 µM WM for 2 h, and 20 µM CCCP was added after 30 min for the final 1.5 h of treatment, and GFP-LC3 puncta visualized as in (B). (D) HeLa cells were starved with HBSS for 4 h as in (B), before 2 h of treatment with 1 µM WM, 20 µM CCCP and/or 100 nM Baf. The cells were lysed in RIPA buffer and the cell lysate was subjected to SDS-PAGE and immunoblotting with anti-LC3 antibody to quantify the endogenous LC3-II to LC3-I ratio. (E) GFP-LC3 HEK293-A cells were starved with HBSS for 4 h before a 2 h incubation in the presence or absence of 1 µM WM in HBSS, with or without 1 µM Oligo and 1 µM AntiA and imaged as in (B). (F) GFP-LC3 HEK-A cells were cultured in DMEM+FCS or starved in HBSS for 2 h in the presence or absence of 5 µM, 10 µM or 20 µM CCCP. Scale bars: 10 µm. N = 3. Error bars indicate SEM, non-significance (ns) and P < 0.05 are annotated.


vacuole of cells incubated with CCCP at any time during the 24 h starvation period (Fig. 4M). In contrast, the proportion of cells incubated without added CCCP and showing red vacuoles increased to over 96% during the same time period, indicating that CCCP inhibits starvation-induced mitophagy in yeast. A similar inhibition of mitophagy with CCCP was observed after induction with rapamycin (Fig. 4G, H, and N). These results indicate that CCCP blocks mitophagy induction downstream of the TOR pathway.

We further assessed whether the CCCP treatment-induced pH-equilibration between the lysosomal lumen and the extracellular medium affects vacuolar degradation. We nitrogen-starved yeast cells for 6 h to label vacuoles with mtRosella (Fig. 4I), then followed degradation of the red fluorophore during CCCP incubation in unbuffered (pH 4.7, Fig. 4K) or neutrally buffered (pH 7.2, Fig. 4L) starvation media. As expected, the proportion of red vacuoles in the neutrally buffered cells remained stable in the presence of CCCP, whereas cells in unbuffered starvation

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Figure 4. CCCP equilibrates yeast vacuolar pH, blocks the initiation of mitophagy and inhibits the degradation of vacuolar content. (A) Mitophagy was assayed in yeast cells expressing mtRosella; a mitochondrially targeted pH-biosensor that fluoresces both red and green within mitochondria (yellow in overlay), and red but not green fluorescent in the acidic vacuole lumen. (B, C, and D) Yeast cells expressing mtRosella were imaged by live-cell fluorescence microscopy (B) without nitrogen starvation, (C) after nitrogen starvation for 24 h, or (D) after nitrogen starvation for 24 h followed by 60 s of incubation with 5 µM CCCP in pH 8.0-buffered 20 mM HEPES. (E, F, and M) Yeast cells expressing mtRosella were imaged after nitrogen starvation for 0, 5, 11 or (E and F) 24 h in the (F and M; dashed line) presence or (E and M; solid line) absence of 20 µM CCCP. (M) The red vacuolar fluorescence of individual cells (n > 300) was scored to determine the percentage of cells undergoing mitophagy. (G, H, and N) Yeast cells expressing mtRosella were imaged after incubation with 2 µM rapamycin for 0 or (G and H) 10 h, either with (H and N; dashed line) or without (G and N; solid line) 20 µM CCCP incubation. (N) The percentage of cells undergoing mitophagy was quantified as in (M). (I–L and O) Yeast cells expressing mtRosella were (I) imaged after 6 h of nitrogen starvation, then re-imaged after incubation for 0.5, 3 and (J, K, and L) 12 h, in either (J) unbuffered starvation medium, (K) unbuffered starvation medium containing 20 µM CCCP, or (L) 20 µM CCCP in HEPES, pH 7.2. (O) The proportion of cells with red vacuolar fluorescence was determined as in (M and N). All images were acquired using the same microscope settings. Scale bars: 10 µm. N = 3. Error bars indicate SEM.

Figure 5. For figure legend, see page 1870.




medium began to lose red vacuolar fluorescence immediately after CCCP addition (Fig. 4O). The results show that in the presence of CCCP, degradation in the vacuole is dependent on the extracellular pH. Mitochondrial remnants persist in PARK2-expressing cells after prolonged exposure to CCCP CCCP is commonly used to stimulate PARK2-mediated mitophagy in mammalian cells.4,28,29 To investigate the effect of CCCP on mitophagy in cells overexpressing PARK2, we cotransfected HeLa cells with expression vectors encoding YFPPARK2 and dsRed2-Mito. We first-tested whether CCCP could promote efficient recruitment of PARK2 to the mitochondrion. As shown in Video S3 a rapid accumulation of PARK2 to the surface of all mitochondria occurred within 1 h of CCCP addition. Cells immunostained for TOMM20 (outer mitochondrial membrane protein) after this treatment, showed colocalization between TOMM20 and dsRed2-Mito (Fig. 5A). After 12 h of CCCP incubation, TOMM20 and dsRed2-Mito colocalized in vesicular structures (~500 nm in diameter) coated with YFPPARK2 and clustered in the perinuclear area (Fig. 5B). Analysis of the immunofluorescence images (using the ImageJ macro outlined in Fig. S1) revealed that TOMM20 levels were greatly reduced in cells overexpressing YFP-PARK2 after 24 h of CCCP incubation (Fig. 5J), consistent with previous reports by Chan et al.28 However, there was no reduction in dsRed2-Mito (Fig. 5H), regardless of different cellular YFP-PARK2 expression levels observed in our experiments (Fig. 5E–I). All labeled TOMM20 remained colocalized with dsRed2-Mito in cells expressing YFP-PARK2, indicating that CCCP had no effect on the mitochondrial localization of dsRed2-Mito. To investigate the degradation of proteins from other compartments we immunostained cells for CYCS (cytochrome c, somatic), COX4I1 (cytochrome c oxidase subunit IV isoform 1) and NDUFAF2/ mimitin [NADH dehydrogenase (ubiquinone) complex I, assembly factor 2], which are located in the inner membrane, intermembrane space and matrix, respectively (Fig. 5K–M; Fig. S2). Cells expressing PARK2 showed reduced levels of each protein, indicating increased degradation of whole mitochondria in PARK2expressing cells. However, in contrast to TOMM20, levels of CYCS, COX4I1 and NDUFAF2 showed a negligible decrease

after 12 or 24 h of CCCP treatment. To test whether TOMM20 degradation is proteasome dependent, we incubated cells with CCCP and monitored the labeling intensity of TOMM20 in the absence and presence of the proteasome inhibitor MG132 (Fig. 5D and N–P; Fig. S3). Degradation of TOMM20 was inhibited in the presence of MG132. These results indicate that degradation by the proteasome and not the lysosome is responsible for CCCP-induced degradation of TOMM20 in the presence of PARK2. To morphologically identify the dsRed2-Mito-positive structures, we employed correlative light and electron microscopy (CLEM). HeLa cells expressing dsRed2-Mito and YFP-PARK2 were incubated with CCCP for 24 h, then imaged using livecell microscopy (Fig. 6A). A single cell expressing both fluorescent constructs was identified (Fig. 6A and B) and processed for transmission electron microscopy (TEM). TEM images of the cell revealed a conspicuous absence of conventional mitochondria. However, spatial alignment between the TEM and optical data revealed that dsRed2-Mito fluorescence colocalized with a set of membrane-bound structures (Fig. 6C–G), but not with multivesicular bodies or lamellar bodies. Although the dsRed2positive structures were not clearly identifiable as mitochondria, they possessed morphological features normally characteristic of mitochondria. The structures frequently appeared to be partially delimited by a discontinuous double membrane, and occasionally contained electron dense inclusions, similar in size and appearance to mitochondrial matrix granules.30 These results suggest that CCCP treatment of PARK2-expressing cells may not result in the complete elimination of all mitochondrial components from the cell, but rather a loss of selected proteins and morphological characteristics typically used to identify them.

Discussion The use of CCCP is increasingly reported in the literature as an inducer for mitophagy and bulk autophagy. Our results raise a number of significant considerations regarding the interpretation of data obtained using CCCP. First, autophagy assays typically rely on steady-state measurements as indicators of autophagic flux. CCCP inhibits degradation in the lysosome resulting in an

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Figure 5 (See previous page). CCCP treatment of cells overexpressing PARK2 results primarily in proteasomal degradation of TOMM20. (A–D) HeLa cells were transfected with dsRed2-Mito and YFP-PARK2 1 d prior to incubation with 20 µM CCCP for (A) 0, (B) 12 or (C) 24 h, or (D) 20 µM CCCP and 10 µM MG132 (proteasome inhibitor) for 24 h, then fixed and processed for imaging of YFP-PARK2, dsRed2-Mito and TOMM20 immunofluorescence by confocal microscopy. (E–M) HeLa cells were transfected with dsRed2-Mito and YFP-PARK2 1 d prior to incubation with 20 µM CCCP for 0, 12 or 24 h, then fixed and processed for immunofluorescence imaging of (E–I) dsRed2-Mito, YFP-PARK2 and one of four endogenous proteins (J–M; TOMM20, CYCS, COX4I1, NDUFAF2). Images of individual cells (> 250, N = 3) were analyzed using the macro outlined in Figure S1, to measure the mean mitochondrial signal intensity of (E–H) dsRed2-Mito, endogenous mitochondrial proteins and the mean cellular signal intensity of YFP-PARK2 for each cell. (E–G) The mitochondrial intensity of dsRed2-Mito in each cell after (E) 0, (F) 12 or (G) 24 h was plotted on a scatter-chart against its mean cellular YFP-PARK2 signal. Cells with YFP-PARK2 fluorescence above a threshold value (E,G, and F; q, 300AU) were classified as “PARK2 expressing”. (H) The mean dsRed2Mito intensity during CCCP treatment was plotted over time for the cells expressing PARK2 (solid line) and non-expressing cells (dashed line). (I) Cells with a mitochondrial DsRed2-Mito fluorescence above a threshold value (E,G, and F; 300AU threshold, t) were classified as “DsRed2 expressing,” and the proportion of cells expressing one, both, or neither construct was charted. (J–M) The mean mitochondrial intensity of (J) TOMM20, (K) CYCS, (L) COX4I1, and (M) NDUFAF2 was plotted over time for the cells expressing PARK2 (solid line) and non-expressing cells (dashed line), using the same criteria as in (H) with the scatter-charts shown in Figure S2. (N–P) HeLa cells were transfected with dsRed2-Mito and YFP-PARK2 1 d prior to incubation for 0, 12, or 24 h with (N) 20 µM CCCP, (O) 20 µM CCCP and 10 µM MG132, or (P) 10 µM MG132. The cells were fixed, labeled and imaged for YFP-PARK2, dsRed2Mito and TOMM20, then analyzed and classified by PARK2 expression using the same criteria as in (H–M) with the scatter-charts shown in Figure S3. Scale bars: 10 µm.

apparent increase in levels of autophagy. Second, CCCP further confounds measurements of autophagic flux by inhibiting the induction of autophagy under certain conditions. Third, mitochondrially targeted dyes relocate to lysosomal compartments upon CCCP treatment. Finally, mitochondrial compartments persist in PARK2-expressing cells after CCCP treatment, but they may not be readily identified due to their altered morphology, which includes the loss of cisternae and partial degradation of the outer membrane. While the association of mitochondrial dyes with other organelles has been described in the literature,31 the targeting of such dyes to lysosomal compartments has not. However, published observations showing vesicular localization of mitochondrial dyes upon CCCP treatment,32-35 are consistent with our data. We tested four mitochondrial dyes, all of which localized

to lysosomal compartments in the presence of CCCP regardless of whether the dye was added before or after CCCP. The only observable differences were in the relative fluorescence intensity and in the kinetics of dye translocation. Of the dyes investigated, MitoTracker FM dyes relocated to the lysosome with the slowest kinetics (data not shown), which is likely due to differences in charge distribution and the number of lipophilic moieties present in the dye molecule. Given the potentiometric properties of the cationic mitochondrial dyes, we suggest that CCCP promotes the formation of a lysosomal membrane potential by dissipating the pH gradient without immediately affecting the gradients associated with other ions, such as chloride. Given the lack of a suitable probe for measuring lysosomal chloride concentrations,19 we suggest that lysosomal uptake of potentiometric dyes during protonophore treatment might be a useful indirect measure of ion




Figure 6. Cells overexpressing PARK2 retain mitochondrial structures after prolonged exposure to CCCP. (A–G) HeLa cells transiently expressing YFPPARK2 and dsRed2-Mito were grown on a polymer film labeled with an alphanumeric reference grid for 1 d, then incubated with 20 µM CCCP for 24 h and stained with 2.5 µg.ml-1 CellMask Deep-Red and 2.5 µM DRAQ5 30 min prior to imaging. (A) Live-cell fluorescence microscopy was used to identify a target cell expressing both YFP-PARK2 and dsRed2-Mito prior to fixation with 4% PFA (cell outline indicated by dashed line). (B) During fixation, 3D optical sections were acquired of the dsRed2-Mito, CellMask and DRAQ5 fluorescence for deconvolution. The cell was relocated and processed for TEM, and the TEM images were spatially aligned to the deconvolved CellMask and DRAQ5 optical data. (C) Two regions of interest (D and F) were selected and (E and G) imaged at higher magnification using TEM. Scale Bars: (A) 10 µm; (B) 5 µm; (C) 2 µm; (D and F) 500 nm; (E and G) 200 nm.

to the literature, we found that CCCP treatment of mammalian cells inhibits starvation-induced autophagy. However, under normal growth conditions we observed a differential effect with numbers of GFP-LC3 puncta being decreased and increased at low and high concentrations of CCCP, respectively. We hypothesized that this outcome may be caused by a discrepancy between the rates at which induction and degradation are inhibited. The inhibition of lysosomal degradation by CCCP is dose-dependent, due to the proportional relationship between concentration and membrane proton conductance.5 However, if the maximum inhibition of induction occurs at low concentrations of CCCP it may not appear to be dose-dependent. This inequality in concentration dependence would result in differential inhibition of autophagy, causing a dose-dependent increase or decrease in the apparent autophagic flux. CCCP may inhibit autophagic induction by a range of mechanisms, including disturbance of pH-sensitive membrane transport or disrupting the cytoskeleton. Investigating the underlying mechanism merits further investigation. It should be noted that while our results highlight significant concerns regarding the application of CCCP to investigate autophagy, they do not necessarily contradict the PINK1-PARK2 model. The mitochondrial remnants we identified by CLEM imaging are remarkably similar in appearance to those observed by Yoshii et al. in Rb1cc1/Fip200 knockout MEFs after 12 h CCCP, and ruptured by proteasomal activity.10 Given the effects of CCCP on lysosomal degradation, our data appear to support a proteasome-dependent clearance mechanism in the presence of CCCP (Fig. S4B). Importantly, we show that the use of AntiA and Oligo together depolarize mitochondria without inhibiting lysosomal degradation. We therefore suggest a combination of AntiA and Oligo to induce mitochondrial depolarization as a viable alternative to the use of CCCP as a suitable trigger for inducing PARK2-mediated mitophagy.45

Materials and Methods Materials Primary rabbit anti-peroxidase (P7899) and anti-LC3B (L7543) antibodies were obtained from Sigma-Aldrich, rabbit antiACTB/β-Actin (4970) and rabbit anti-COX4I1 (4850P) from Cell Signalling Technologies, mouse anti-CYCS (556433) from BD Biosciences, and rabbit anti-TOMM20 (sc-11415) from Santa Cruz Biotechnology. Secondary HRP-conjugated donkey antirabbit IgG (RPN4301) was from GE Healthcare and Alexa Fluor 647-conjugated donkey anti-rabbit (A21428) from Invitrogen. Bafilomycin A1 (BIA-B1110) was purchased from BioAustralis, unconjugated HRP (01-2001) and HBSS (14025-092) from Invitrogen, and wortmannin (W1628), oligomycin (O4879), antimycin A (A8674), MG132 (C2211), and CCCP (C2759) were obtained from Sigma-Aldrich. For live-cell microscopy experiments TMRM (T668), DiOC6(3) (D273), MitoTracker Red FM (M22425), MitoTracker Green FM (M7514), LysoTracker Red DND-99 (L7528), LysoTracker Green DND-26 (L7526) and Cell-Mask Deep Red (C10046) from Invitrogen, and DRAQ5 (DR50050) from BioStatus was used.

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gradients between the lysosome and cytoplasm, especially if used in conjunction with other ionophores. The retargeting of mitochondrial dyes is particularly important in the context of mitophagy research. Our observations are highly reminiscent of results reported by Ding et al. that describe changes in the morphology of mitochondria and their “acidification” after treatment of cells with CCCP.12 Our results suggest that the apparent “acidification of mitochondria” after CCCP treatment is artifactual, and more likely caused by translocation of mitochondrial dyes to the lysosome. Surprisingly, the discrepancy between fluorescent proteins and mitochondrial dyes is also demonstrated in the paper by Ding et al., however, retargeting of the mitochondrial dyes was not taken into consideration in that report. Our results show that caution should be exercised when organelle-“specific” fluorescent dyes are used in conjunction with compounds or conditions capable of nonspecifically affecting the physical, structural or chemical properties of cellular membranes. The ability of CCCP to equilibrate lysosomal pH is unsurprising, as the kinetic mechanism of proton translocation by CCCP is not specific to mitochondria.5 Indeed, a number of reports describe the effects of CCCP and similar protonophores not only on lysosomes,13 but also the Golgi complex,36,37 and microtubules.38 The effects of CCCP on the lysosome and autophagy have yet to be adequately addressed in the literature, with the exception of Ding et al. who reported no effect of CCCP on lysosomal degradation.20 We observed significant differences between the inhibitory effects of CCCP and Baf. These differences can be explained by the different mechanisms by which each compound affects the lysosome. Inhibition of the V-ATPase activity by Baf will affect lysosomal pH more slowly than the rapid equilibration of protons by CCCP. This discrepancy is not dissimilar to the divergent effects of Baf and chloroquine on endosomal trafficking.39,40 Interestingly, Chan et al. have previously reported an increase in V-ATPase levels in cells treated with CCCP, which may represent a compensatory response to CCCP.28 Even though WM inhibits most autophagic pathways including a noncanonical ATG5-independent pathway,41 several instances of PIK3CAindependent autophagy have been described including induction by glucose deprivation and photo-irradiation.42,43 However, our data show that WM completely blocks any activating effect of CCCP on autophagy. The inability of CCCP to induce mitophagy in yeast cells has been attributed to the absence of PINK1 and PARK2 homologs in these cells.24,44 We found that CCCP inhibits mitophagy in yeast cells, a different conclusion to that reported by Mendl et al., that might be explained by differences in the assays used.23 Our assay monitors delivery of a protein located in the mitochondrial matrix to the lysosome by fluorescence microscopy, whereas Mendl et al. measured the activation of a mitochondrially-targeted Pho8 by the vacuolar hydrolase Pep4. The fact that CCCP causes different effects in the two assays might be a further indication of the complexity of changes caused by equilibrating the pH across all intracellular compartments with the extracellular medium. Our results in yeast indicate that the inhibition mechanism of CCCP is downstream of the TOR pathway. Contrary

mitochondria using the citrate synthase mitochondrial targeting sequence and encoded on the single copy plasmid pRS413 under expression control of the ADH1 short variant promoter. Details of the Rosella-based mitophagy assay have been published elsewhere,25,26 and are briefly outlined here with some modifications. For live-cell fluorescence microscopy, cells were grown to mid-log phase in synthetic complete medium containing 2% (v/v) ethanol as the carbon source. Cells were collected by centrifugation, rinsed with water and resuspended to OD600 = 0.4 in nitrogen starvation medium [2% (v/v) ethanol in 0.17% (w/v) yeast nitrogen base without amino acids or ammonium sulfate (BD Difco, 233520)]. Cells were cultured under nitrogen starvation conditions with or without added 20 µM CCCP for the time periods indicated. Where indicated, cells were starved for 6 h prior to addition of 20 µM CCCP, and other samples buffered to pH 7.2 using 20 mM HEPES-KOH as indicated. Cells were prepared for imaging by adhesion as a monolayer to a 96-well CellCarrier (Perkin Elmer, 6005558) coated with concanavalin A (SigmaAldrich, C5275). Microscopy was immediately performed at room temperature on an Olympus FV500 inverted confocal microscope with a 60× water immersion objective (UPlanApo, NA 1.20; Olympus). All photomultiplier settings remained unchanged throughout each experiment. Individual cells (N > 300) from each image were scored for red vacuolar fluorescence to determine the percentage of cells exhibiting mitophagy. CLEM HeLa cells were grown on TOPAS 6013 film marked with an alphanumeric reference grid, then transfected with both YFPPARK2 and dsRed2-Mito 24 h prior to the experiment. The cells were then incubated with 20 µM CCCP for 24 h, and stained with 2.5 µg.ml−1 CellMask Deep-Red (Invitrogen, C10046) and 2.5 µM DRAQ5 (BioStatus, DR50050) 30 min prior to imaging via live-cell confocal microscopy on an Olympus FluoView FV1000 inverted confocal microscope, using a 60× objective lens (PLAPO, NA 1.00; Olympus). A cell expressing both YFP-PARK2 and dsRed2-Mito was identified prior to fixation for 1 h with 4% paraformaldehyde in 0.1 M phosphate buffer. Three-dimensional image data was acquired for deconvolution before post-fixation for 1 h with 2.5% glutaraldehyde in 0.1 M phosphate buffer. The sample was prepared for standard TEM including post-fixation with 1% aqueous osmium tetroxide, en-bloc staining with 2% aqueous uranyl acetate, and embedding in Procure-Araldite resin (ProSciTech, C039) after standard dehydration. The cell was relocated and sectioned using an Ultracut UCT ultramicrotome (Leica) to cut 70-nm ultrathin sections, which were stained with uranyl acetate for 5 min and lead citrate for 3 min before images were acquired on a Hitachi 7500 TEM. The TEM images were aligned to the deconvolved fluorescence data. Statistics and analysis Statistical analysis was performed using Mathematica.47 Immunoblots were analyzed using the densitometry package in ImageJ.48 CLEM optical data was deconvolved using Huygens Professional,49 and was spatially aligned in the GNU Image Manipulation Program.50 Immunofluorescence images were characterized using a macro written for ImageJ, which is summarized in Figure S2.




Mammalian cell culture GFP-LC3 HEK293-A cells were generously provided by Sharon Tooze (Cancer Research UK). All cells were cultured in DMEM containing 10% FCS. HeLa cells were transiently transfected with pDsRed2-Mito (Clontech, 632421) and YFPPARK2 (Addgene, 23955) 24 h prior to the experiment using Lipofectamine 2000 (Invitrogen, 11668) according to the manufacturer’s protocol. HRP degradation assay and autophagosome clearance assay To measure HRP degradation, HeLa cells were loaded with HRP by incubation with 0.25 mg.ml−1 unconjugated HRP in serum-free DMEM for 2 h.46 The cells were rinsed 8 times with full-serum DMEM for 5 min per rinsing step. The cells were then cultured for up to 24 h in the presence or absence of 20 µM CCCP, followed by lysis using RIPA buffer (1% NP-40, 0.1% SDS, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 1 mM EDTA) containing complete protease inhibitors (Roche Applied Science, 05892953001). The effect of CCCP on autophagosomal clearance was analyzed by immunoblotting of HeLa cell lysates. After induction of autophagy by starvation in HBSS for 4 h, cells were treated for an additional 2 h with DMSO alone, 1 µM WM, 20 µM CCCP, 100 nM Baf, or a combination of the drugs. WM was refreshed after 1 h of treatment. The samples were lysed using RIPA buffer containing protease inhibitors. Proteins were separated by SDSPAGE, followed by western blot transfer to a PVDF membrane. Primary antibody incubations for LC3 and ACTB were followed by secondary HRP-conjugated anti-rabbit IgG and visualized using SuperSignal West Pico chemiluminescent substrate (Pierce, 34079). Optical microscopy Fixed cell confocal microscopy was performed on GFP-LC3 HEK293-A and HeLa cells starved with HBSS for 6 h with drug treatment beginning after 4 h of starvation. The samples were treated with or without 1 µM WM, 20 µM CCCP, or both. The samples were fixed with 4% PFA and the GFP fluorescence was observed using an Olympus FluoView FV1000 inverted confocal microscope with a 60× objective lens (PLAPO, NA 1.00; Olympus). For live-cell confocal microscopy, HeLa cells were grown in 35-mm FluoroDishes (World Precision Instruments) for at least 18 h prior to imaging, with media substitution to a phenol redfree equivalent 1 h prior to imaging. Samples were stained prior to imaging for 30 min with either 20 nM DiOC6(3), 500 nM TMRM, 100 nM MTR, or MTG; 100 nM LTR (DND-99) or LTG (DND-26); or a combination thereof. Live-cell time series data were acquired on an Olympus FluoView FV1000 inverted confocal microscope, using a 60× objective lens (PLAPO, NA 1.00; Olympus) and equipped with a thermally regulated incubator hood with CO2 controller (Clearstate Solutions). Fluorophores were excited using 488 nm, 543 nm and 633 nm laser lines. Time-series data were acquired at 0.2 fps for 50 s before CCCP treatment, and for 10 min after treatment with 20 µM CCCP (unless indicated otherwise). Yeast work was conducted using Saccharomyces cerevisiae strain BY4741 expressing the biosensor mtRosella, which was targeted to

The authors declare that there are no conflicts of interest.

microscopy, Rod Devenish for critically reading the manuscript, Sharon Tooze for the gift of GFP-LC3 HEK-A cells, and Mike Ryan and Kirstin Elgass for providing antibodies. This work was supported by NHMRC project grant funding to GR and ARC discovery project funding to MP.

Acknowledgments

Supplemental Materials

Disclosure of Potential Conflicts of Interest

We would like to thank Stephen Firth, Alex Fulcher, and July Callaghan from Monash Micro Imaging for help with


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Ammonium ion interferes with the lysosomal degradation of glycosaminoglycans in cultures of human glial cells.

Retromer Ensures the Degradation of Autophagic Cargo by Maintaining Lysosome Function in Drosophila.

Lysine suppresses myofibrillar protein degradation by regulating the autophagic-lysosomal system through phosphorylation of Akt in C2C12 cells.

Lysine suppresses protein degradation through autophagic-lysosomal system in C2C12 myotubes.

lysosomal dysfunction in Alzheimer's disease.

The autophagic lysosomal system in outflow pathway physiology and pathophysiology.

p97 Proteins Are Targets of Autophagic-Lysosomal Degradation.

Lysosomal Regulation of mTORC1 by Amino Acids in Mammalian Cells.

The autophagic- lysosomal pathway determines the fate of glial cells under manganese- induced oxidative stress conditions.

Transfer of yeast artificial chromosomes from yeast to mammalian cells.

Silver Nanoparticle-Induced Autophagic-Lysosomal Disruption and NLRP3-Inflammasome Activation in HepG2 Cells Is Size-Dependent.

Septin 9 interacts with kinesin KIF17 and interferes with the mechanism of NMDA receptor cargo binding and transport.

Massively parallel delivery of large cargo into mammalian cells with light pulses.

Autophagic degradation of peroxisomes in mammals.

The role of autophagic degradation in the heart.

Fucosylation of LAMP-1 and LAMP-2 by FUT1 correlates with lysosomal positioning and autophagic flux of breast cancer cells.

Infection with Usutu virus induces an autophagic response in mammalian cells.

proteasomal degradation.

Artesunate induces cell death in human cancer cells via enhancing lysosomal function and lysosomal degradation of ferritin.

Differential degradation of messenger RNAs in mammalian cells.

Conessine Interferes with Oxidative Stress-Induced C2C12 Myoblast Cell Death through Inhibition of Autophagic Flux.

Peroxin Pex14p is the key component for coordinated autophagic degradation of mammalian peroxisomes by direct binding to LC3-II.

Overexpression of Aurora-C interferes with the spindle checkpoint by promoting the degradation of Aurora-B.

Hepatitis C virus present in the sera of infected patients interferes with the autophagic process of monocytes impairing their in-vitro differentiation into dendritic cells.